Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Protein fold recognition score functions: Unusual construction strategies

We describe two ways of optimizing score functions for protein sequence to structure threading. The first method adjusts parameters to improve sequence to structure alignment. The second adjusts parameters so as to improve a score function's ability to rank alignments calculated in the first score function. Unlike those functions known as knowledge-based force fields, the resulting parameter sets do not rely on Boltzmann statistics, have no claim to representing free energies and are purely constructions for recognizing protein folds. The methods give a small improvement, but suggest that functions can be profitably optimized for very specific aspects of protein fold recognition, Proteins 1999;36:454-461. (C) 1999 Wiley-Liss, Inc.

Protein sequence threading, the alignment problem, and a two-step strategy

Conventionally, protein structure prediction via threading relies on some nonoptimal method to align a protein sequence to each member of a library of known structures. We show how a score function (force field) can be modified so as to allow the direct application of a dynamic programming algorithm to the problem. This involves an approximation whose damage can be minimized by an optimization process during score function parameter determination. The method is compared to sequence to structure alignments using a more conventional pair-wise score function and the frozen approximation. The new method produces results comparable to the frozen approximation, but is faster and has fewer adjustable parameters. It is also free of memory of the template's original amino acid sequence, and does not suffer from a problem of nonconvergence, which can be shown to occur with the frozen approximation. Alignments generated by the simplified score function can then be ranked using a second score function with the approximations removed. (C) 1999 John Wiley & Sons, Inc.

Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods, These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA, To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters, In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [H-3]N-OH-IQ to DNA, ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and similar to 50% of that seen with acetyl coenzyme A (AcCoA), In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites, With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein, The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: Correlations with fetal growth

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases of noninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFSP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-5 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGK at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa - 2. Three-dimensional solution structure

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D H-1 NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and mu O-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18 +/- 0.05 Angstrom for the backbone atoms and 1.39 +/- 0.33 Angstrom for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.

Feed intake and liveweight responses to nitrogen and/or protein supplements by steers of Bos taurus, Bos indicus and Bos taurus x Bos indicus breed types offered a low quality grass hay

Thirty steers were used in two pen experiments (Expts 1 and 2). and 27 of these in a third (Expt 3), to quantify their responses of hay intake, rumen ammonia nitrogen (RAN) concentrations, and liveweight to inputs of rumen soluble nitrogen (urea) and rumen undegradable protein (formaldehyde-treated casein; F-casein) when added to a basal diet of low quality hays. The hays were made From unimproved native pastures typical of those grazed by cattle in the subtropics of Australia and contained 7.8 g N/kg dry matter (DM) with coefficient of organic matter digestibility of 0.503 in Expts 1 and 2, and 5.2 g N/kg DM with a digestibility range from 0.385 to 0.448 in Expt 3. The steers (15 months old) were either Brahman (B), Hereford (H) or the F-1 Brahman x Hereford (BH) cross. Steers were offered supplementary minerals with the hays in each experiment. In Expt 1 (35 days) urea was sprayed on part of the hay, allowing for daily urea intakes (g/steer) of either 0, 5, 11, 16 or 26. In Expt 2 (42 days), F-casein was offered daily (g/steer) at either 0, 75, 150, 225 or 300 and in Expt 3 (56 days) discrete offerings were made of soluble casein (225 g/day), of urea (18 g/day) + F-casein (225 g/day) or of nil. There were significant linear effects of urea intake upon hay intake and liveweight change of steers. However, B steers had smaller increases in intake and liveweight change than did H steers, and B steers did not have a linear increase in RAN concentrations with increasing urea intake as did H and SH steers. In Expt 2 there were significant linear effects of F-casein supplements on hay intake and liveweight change of steers and a significant improvement in their feed conversion ratio (i.e. DM intake:liveweight change). The B steers did not differ from H and BH steers in liveweight change but had significantly lower hay intakes and non-significantly smaller increases in RAN with increasing F-casein intake. In Expt 3, hay intake of the steers increased with soluble casein (by 16.8 %) and with urea + F-casein (24.5 %). Only steers given urea + F-casein had a high RAN concentration (94 mg/l) and a high liveweight gain. The B steers had a liveweight loss and a lower hay intake than H or BH steers in Expt 3 but a higher RAN concentration. These studies have indicated the importance of the form and quantity of additional N required by cattle of differing breed types to optimize their feed intake and liveweight gain when offered low-N, low-digestible hays.

Multiple conformational epitopes are recognized by natural and induced immunity to the E7 protein of human papilloma virus type 16 in man

The reactivity of sera from patients with cervical cancer with the E7 protein of human papilloma virus type 16 (HPV16) was estimated using a novel non-radioactive immunoprecipitation assay and four established protein-and peptide-based immunoassays. Six of 14 sera from patients with cervical cancer and 1 of 10 sera from healthy laboratory staff showed repeated reactivity with E7 in at least one assay. Four of the 7 reactive sera were consistently reactive in more than one assay, but only one was reactive in all four assays. Following immunization with E7, 2 of 5 patients with cervical cancer had increased E7-specific reactivity, measurable in one or more assays. No single assay was particularly sensitive for E7 reactivity, or predictive of cervical cancer. Mapping of E7 reactivity to specific E7 peptides was unsuccessful, suggesting that natural or induced E7 reactivity in human serum is commonly directed to conformational epitopes of E7, These results suggest that each assay employed with is study measures a different aspect of E7 reactivity, and that various reactivities to E7 may manifest following HPV infection or immunization. This finding is of significance for monitoring of E7 immunotherapy and for serological screening for cervical cancer. Copyright (C) 2000 S.Karger, AG. Basel.

Characterization of peroxisome proliferator-activated receptor alpha in normal rat mammary gland and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary gland tumors from rats fed high and low fat diets

Normal Sprague-Dau ley rat mammary gland epithelial cells and mammary gland carcinomas induced by 2-amino-1 -methyl-6-phenylimidazo[4,5-b]pyridine, a carcinogen found in the diet, were examined for the expression of peroxisome proliferator-activated receptor alpha (PPAR alpha). PPAR alpha mRNA and protein was detected in normal and tumor tissue by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. By quantitative RT-PCR, carcinomas had a 12-fold higher expression than control mammary glands, a statistically significant difference. PPAR alpha expression was examined in carcinomas and normal tissues from rats on high fat (23.5/% corn oil) and low fat (5% corn oil) diets. Although neither carcinomas, nor control tissues showed statistically significant differences between the two diet groups, PPAR alpha expression was the highest in carcinomas from rats on the high fat diet. The expression of PPAR alpha in normal mammary gland and its significant elevation in mammary gland carcinomas raises the possibility of its involvement in mammary gland physiology and pathophysiology. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

Crystal structure of a heptameric Sm-like protein complex from archaea: Implications for the structure and evolution of snRNPs

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautrophicum at 2.0 Angstrom resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta -strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta -sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis. (C) 2001 Academic Press.

Truncation of the GABA(A)-receptor gamma 2 subunit in a family with generalized epilepsy with febrile seizures plus

Recent findings from studies of two families have shown that mutations in the GABA(A)-receptor gamma2 subunit are associated with generalized epilepsies and febrile seizures. Here we describe a family that has generalized epilepsy with febrile seizures plus (GEFS(+)), including an individual with severe myoclonic epilepsy of infancy, in whom a third GABA(A)-receptor gamma2-subunit mutation was found. This mutation lies in the intracellular loop between the third and fourth transmembrane domains of the GABA(A)-receptor gamma2 subunit and introduces a premature stop codon at Q351 in the mature protein. GABA sensitivity in Xenopus laevis oocytes expressing the mutant gamma2(Q351X) subunit is completely abolished, and fluorescent-microscopy studies have shown that receptors containing GFP-labeled gamma2(Q351X) protein are retained in the lumen of the endoplasmic reticulum. This finding reinforces the involvement of GABA(A) receptors in epilepsy.

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp PCC6803 DnaB mini-intein

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures similar to14 degreesC higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH2 and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (DeltaDeltaG approximate to 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.

Myb-binding protein 1a augments AhR-dependent gene expression

We have studied the mechanism by which an acidic domain (amino acids 515-583) of the aromatic hydrocarbon receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein la, whereas a mutant domain (F542A, 1569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein la into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein la is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein la associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein la to increase AhR-dependent gene expression.

Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cells

A flow chamber was used to impart a steady laminar shear stress on a recombinant Chinese hamster ovary (CHO) cell line expressing human growth hormone (hGH). The cells were subjected to shear stress ranging from 0.005 to 0.80 N m(-2). The effect of shear stress on the cell specific glucose uptake, cell specific hGH, and lactate productivity rates were calculated. No morphological changes to the cells were observed over the range of shear stresses examined. When the cells were subjected to 0.10 N m(-2) shear in protein-free media without Pluronic F-68, recombinant protein production ceased with no change in cell morphology, whereas control cultures were expressing hGH at 0.35 mug/10(6) cells/h. Upon addition of the shear protectants, Pluronic F-68 (0.2% [w/v]) or fetal bovine serum (1.0% [v/v] FBS), the productivity of the cells was restored. The effect of increasing shear stress on the cells in protein-free medium containing Pluronic F-68 was also investigated. Cell specific metabolic rates were calculated for cells under shear stress and for no-shear control cultures performed in parallel, with shear stress rates expressed as a percentage of those obtained for control cultures. Upon increasing shear from 0.005 to 0.80 N m(-2), the cell specific hGH productivity decreased from 100% at 0.005 N m(-2) to 49% at 0.80 N m(-2) relative to the no-shear control. A concurrent increase in the glucose uptake rate from 115% at 0.01 N m(-2) to 142% at 0.80 N m(-2), and decreased lactate productivity from 92% to 50%, revealed a change in the yield of products from glucose compared with the static control. It was shown that shear stress, at sublytic levels in medium containing Pluronic F-68, could decrease hGH specific productivity. (C) 2002 Wiley Periodicals, Inc.